Quantitative determination of aflatoxin by high performance liquid chromatography in wheat silos in Golestan province, north of Iran

Background: Aflatoxins are the most common mycotoxins that contaminate crops. They are produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. Wheat (Tricitumaestivum) is one of the most important staple foods used in Iran, and the environmental conditions in the north of Iran are favorable to fungal growth. This study was designed in order to determine the aflatoxin concentration in wheat samples from silos in Golestan Province north of Iran. Methods: Samples were collected from three silos of Golestan province. First, aflatoxins were isolated using immu-noaffinity chromatography. Then the aflatoxin concentrations were determined by High performance liquid chroma-tography (HPLC) method and fluorescence detector. Results: Ten out of 34 samples (29.4 of samples) were contaminated by aflatoxins.No concentration was found above permitted aflatoxin levels in Iran (15 ng/g). In one sample (2.9), aflatoxin B1 was seen over the permissible limits in Iran. The highest level found in samples for total aflatoxin, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatox-in G2 were 7.08 ng/g, 6.91 ng/g, 0.29 ng/g, 1.37 ng/g and 0.23 ng/g, respectively. No correlation was found between humidity levels in wheat samples contained aflatoxin and wheat samples without aflatoxin. Conclusion: Despite the total aflatoxins determined in samples were below the permissible limits in Iran, the 29 aflatoxin contamination rate can negatively affect health factors and it should not be neglected. So, it is predictable that if the storage duration of samples increases, the aflatoxin contamination levels will increase. © 2015, Iranian Journal of Public Health. All rights reserved

Aflatoxin contamination in wheat flour samples from Golestan province, Northeast of Iran

Background: Due to the high toxicity of aflatoxin and its effects on public health, determination of aflatoxin level in Wheat flour samples in the Golestan province, north of Iran was investigated. To examine the effect of seasonal changes, summer and winter sampling was performed with standard sampling methods. Methods: A total of 200 flour samples were collected from 25 factories. HPLC method with immunoaffinity chromatography was used to measure aflatoxin types (G2, G1, B2 and B1). Statistical analysis was performed by the Pearson correlation test, One-way ANOVA and multivariate regression analysis. Results: Mean total aflatoxin levels of samples were 0.82 and 1.99 ng/g in summer and winter, respectively. Aflatoxin B1 levels were detected in 3.1%, 7.4% over permissible limits by worldwide regulations in samples collected in summer and winter, respectively. Aflatoxins in winter were higher than summer. The highest frequency of aflatoxin contamination in winter was B2 (98%) and in summer G1 (51%). The relationship between humidity and rate of aflatoxin B1 and total aflatoxin was significant in winter. Results of multivariate regression were showed the strongest relationship with humidity and aflatoxin level. Despite the contamination of flour samples, there was no contamination higher than the standard limit of Iran Standard Institute. But it was significantly higher than similar studies from other regions. Conclusions: Therefore, with regard to negative impacts of aflatoxin on health, aflatoxin contamination should be considered in future programs. Decrease of aflatoxin contamination may be made practical through reducing wheat storage duration and controlling humidity

Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature

A microarray analysis was performed to study the effect of varying combinations of water activity and temperature on the activation of aflatoxin biosynthesis genes in Aspergillus flavus grown on YES medium. Generally A. flavus showed expression of the aflatoxin biosynthetic genes at all parameter combinations tested. Certain combinations of aw and temperature, especially combinations which imposed stress on the fungus resulted in a significant reduction of the growth rate. At these conditions induction of the whole aflatoxin biosynthesis gene cluster occurred, however the produced aflatoxin B1 was low. At all other combinations (25 °C/0.95 and 0.99; 30 °C/0.95 and 0.99; 35 °C/0.95 and 0.99) a reduced basal level of cluster gene expression occurred. At these combinations a high growth rate was obtained as well as high aflatoxin production. When single genes were compared, two groups with different expression profiles in relation to water activity/temperature combinations occurred. These two groups were co-ordinately localized within the aflatoxin gene cluster. The ratio of aflR/aflJ expression was correlated with increased aflatoxin biosynthesis

Determination of aflatoxin M1 levels in 1 white cheese samples by ELISA in Gilan province, Iran

Aflatoxin M1 (AFM1) in milk and milk products is considered to pose certain hygienic risks for human health. These metabolites are not destroyed during the pasteurization and heating process. This study was undertaken to determine the presence and levels of aflatoxin M1 (AFM1) in Iranian white cheese consumed in Gilan province (Northern Iran). A total of 90 cheese samples was randomly obtained from retail outlets. ELISA technique was used to determine the presence and the level of AFM1. In 78 of the 90 cheese samples examined (86.66%), the presence of AFM1 was detected in concentrations between 7.2 - 413ng/l. The mean level of AFM1 in positive samples was 151.97 ng/l. AFM1 levels in 21 samples (23.33%) were higher than the maximum tolerance limit (250 ng/l) accepted by the European countries. Aflatoxin high concentration in milk and milk products cause widespread negative impact on public health and demonstrate considerable economic losses for producers. Therefore, it is necessary to establish strategies for reducing aflatoxin levels in animal feed and milk products. © IDOSI Publications, 2012

Characterisation of a putative dothistromin biosynthetic cluster : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University, Palmerston North, New Zealand

The fungus Dothistroma pini is a key pathogen in New Zealand (and international) softwood plantations, most notably P. radiata. The mycotoxin dothistromin produced by this saprophytic fungus is believed to play a major role in its pathogenesis. Dothistromin shares functional groups and pathway intermediates with those of sterigmatocystin and aflatoxin, secondary metabolites of Aspergillus sp. As the sterigmatocystin and aflatoxin biosynthetic pathways are characterised this provided us with a model pathway and potential probes for the isolation of dothistromin genes. The verl gene is critical to the completion of aflatoxin biosynthesis in Aspergillus sp. as its disruption prevented the synthesis of aflatoxin. Assuming similar enzymes act in the dothistromin biosynthetic pathway a probe for ver1 was obtained and used to probe a D. pini genomic library. This led to the isolation of two lambda clones named λCGV1 and λCGV2 (Gillman 1996). A second library screen was completed using an aflatoxin polyketide synthase (PKS) probe and led to the isolation of the lambda clone λBMKSA (Morgan 1997). The λCGV1 clone has been studied in detail and shown to contain a gene similar to aflatoxin ver1 (named dkr1) and other potential dothistromin biosynthetic genes (Monahan 1998). This study looks in greater detail at the lambda clones λCGV2 and λBMKSA and determines whether they contain putative dothistromin biosynthetic genes and are part of the anticipated gene cluster. In this project the lambda clone λCGV2 was partially characterised which revealed that the other potential ver gene showed a greater similarity to the melanin biosynthetic gene phn than to the aflatoxin gene ver-1. This implied that the clone was unlikely to contain dothistromin biosynthetic genes so no further sequence was generated. However, a partial restriction map was constructed. The other lambda clone, λBMKSA was then further characterised. Double stranded sequence of the putative pks gene region was completed. The remainder of the lambda clone was subcloned and exploratory sequence revealed a gene with high similarity to stcW. The next stage was to determine how the three lambda clones were related. This was approached by probing genomic Southern blots with the ends of the lambda clones to determine the presence of commonly hybridised fragments. The presence of common fragments suggests that the three clones are very close together in the genome, although the evidence which links λCGV2 and λBMKSA is stronger than the evidence that links λCGV2 and λCGV1. This is the first evidence that the three lambda clones isolated using aflatoxin probes are close together in the genome of D. pini. The genes present on these lambda clones show a high degree of similarity to their aflatoxin counterparts and could potentially contain a dothistriomin biosynthetic cluster

High-performance liquid chromatography and Enzyme-Linked Immunosorbent Assay techniques for detection and quantification of aflatoxin B1 in feed samples: a comparative study.

ObjectiveComparison was done between high-performance liquid chromatography (HPLC) and a competitive enzyme-linked immunosorbent assay (ELISA) for detection and quantification of aflatoxin B1 (AFB1) in feed samples. The two procedures were standardized and validated before the actual experiment. Five concentrations (0, 5, 10, 20 and 30 ppb) of feed samples were used for both methods. For the HPLC technique, the samples were extracted in acetonitrile/water (90/10) solution, cleaned-up using solid phase extraction (SPE) column, and derivatized by water/trifluoroacetic acid/glacial acetic acid (35/10/5) solution before instrument analysis. The samples were extracted in 70% methanol for the ELISA technique.ResultsThe two tests showed very strong linearity with correlation coefficient value of > 0.99 using standard solutions. The mean recovery rate was 92.42% (with relative standard deviation (RSD) of 5.97) and 75.64% (RSD = 34.88) for HPLC and ELISA, respectively. There was no statistically significant difference in recovery rate between the two methods. There was a positive correlation (r = 0.84) between them which indicated that the two techniques can be used to detect and quantify aflatoxin B1 in feed samples. However, there were variations among replicates for the ELISA method, which shows that this method is more applicable for screening purposes

Review of mycotoxin reduction in food and feed: from prevention in the field to detoxification by adsorption or transformation

Mycotoxins are secondary metabolites present worldwide in agricultural commodities and produced by filamentous fungi that cause a toxic response (mycotoxicosis) when ingested by animals. Prevention of mycotoxicoses includes pre- and post-harvest strategies. The best way to reduce the mycotoxin content in food and feed is the prevention of mycotoxin formation in the field, but this is often not sufficient, so other methods are needed. To decontaminate and/or detoxify mycotoxin-contaminated food and feed, the most prevalent approach in the feed industry is the inclusion of sorbent materials in the feed thus obtaining more or less selective removal of toxins by adsorption during passage through the gastrointestinal tract. Another reliable approach is to add enzymes or microorganisms capable of detoxifying some mycotoxins. Through a comprehensive review of published reports on the strategies for mycotoxin removal, this present work aims to update our understanding of mycotoxin removal. It provides an insight into the detoxification of mycotoxin present in food and feed. In the future, more emphasis needs to be placed on adsorption of mycotoxins in the gastrointestinal tract. Concerning the enzymatic transformation of mycotoxins, further efforts are required in understanding detoxification reactions, the toxicity of transformation products and in the characterization of enzymes responsible for transformations

Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach

Produced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation